WPI NO Microsensors User manual

Brand: WPI

Model: NO Microsensors

Type: User manual

Instrumenting scientiﬁc ideas

WORLD

PRECISION

INSTRUMENTS

NO Microsensors

ISO-NOPF100, ISO-NOPF200, ISO-NOPF-200-L10, ISO-NOP70L,

ISO-NOP3005, ISO-NOP3020 ISO-NOP30-L, ISO-NOP007, ISO-NOPNM

Serial No._____________________

www.wpiinc.com

INSTRUCTION MANUAL

051316

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World Precision Instruments i

may be reproduced or translated into any language, in any form, without prior written permission of

World Precision Instruments, Inc.

CONTENTS

ABOUT THIS MANUAL ................................................................................................................... 1

INTRODUCTION .............................................................................................................................. 1

Notes and Warnings ................................................................................................................. 2

INSTRUMENT DESCRIPTION ........................................................................................................ 3

Parts List ...................................................................................................................................... 3

Unpacking ................................................................................................................................... 3

OPERATING INSTRUCTIONS ......................................................................................................... 4

Environmental Inuences ....................................................................................................... 4

Temperature ......................................................................................................................... 4

Electrical Interference ...................................................................................................... 4

Attaching the Sensor to the Microsensor Handle ............................................................ 4

Polarizing the Sensor ............................................................................................................... 5

Calibrating the Sensor ............................................................................................................ 6

Understanding SNAP ............................................................................................................... 6

Method 1: SNAP using CuCl .............................................................................................. 6

Method 2: SNAP using CuCl2 ....................................................................................................... 8

Method 3: NO Standards Prepared with NO Gas ....................................................... 9

INSTRUMENT MAINTENANCE .................................................................................................... 12

Storing the Sensor .................................................................................................................. 12

Cleaning the Sensor ...............................................................................................................12

ACCESSORIES.................................................................................................................................12

TROUBLESHOOTING ...................................................................................................................13

SPECIFICATIONS ............................................................................................................................14

APPENDIX A: PREPARING STOCK SOLUTION .........................................................................15

100mM Standard SNAP Solution ........................................................................................15

APPENDIX B: CONSTRUCTING A CALIBRATION CURVE .......................................................16

Predicting the Level of Detectable NO CuCl2 ...........................................................................16

Example for Creating a Calibration Curve .........................................................................17

INDEX ............................................................................................................................................... 21

WARRANTY .....................................................................................................................................23

Claims and Returns ................................................................................................................23

Repairs ....................................................................................................................................... 23

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ABOUT THIS MANUAL

The following symbols are used in this guide:

This symbol indicates a CAUTION. Cautions warn against actions that can

cause damage to equipment. Please read these carefully.

This symbol indicates a WARNING. Warnings alert you to actions that can

cause personal injury or pose a physical threat. Please read these carefully.

NOTES and TIPS contain helpful information.

INTRODUCTION

The NO Microsensors are available in a variety of tip diameters and lengths

for a range of applications. These microsensors incorporate WPI’s proprietary

combination electrode technology whereby the composite graphite nitric oxide

sensing element and separate reference electrode are encased within a single

sensor design.

• Used for in vivo applications, the ISO-NOPF sensors are available in 100µm (WPI

#ISO-NOPF100) and 200µm (WPI #ISO-NOPF200) diameters. They were created

to be exible and are the most durable of all the WPI’s nitric oxide sensors. The

ISO-NOPF is also available with a hypodermic sheath (WPI #ISO-NOPF100H

or ISO-NOPF200H). These sensors are sold in 1-5mm lengths of whole 1mm

increments (1mm, 2mm, 3mm, 4mm, 5mm).

• For use with a tissue bath or in monolayer-style cell culture studies, the ISO-

NOPF200 is available in an L-shape (#ISO-NOPF200-L10), and it is 10mm long.

The L-shaped version is also available in 70mm (WPI #ISO-NOP-70-L) or 30mm

(WPI #ISO-NOP30-L) diameters, both 3mm long.

• When working with microvessels, three options are available. The ISO-NOP007

is 7mm in diameter and 2mm long. The ISO-NOP3005 is 30mm in diameter and

0.5mm long. And, the ISO-NOP3020 is 30mm in diameter and 2mm long.

• For single cell applications, the conical tip of the ISO--NOPNM is only 100mm and

the sensor tip is 150mm long.

Fig. 1 (on the next page) shows a labeled drawing of the carbon ber microsensors.

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2 mm

A or B

Carbon ﬁber with hydrophobic

gas permeable coating

Hydrophobic

gas permeable

coating

Stainless steel handle

Shielded cable

to ISO-NO meter

A = 2 mm tip length

B = 0.5 mm tip length

Flexible

sensor body

Diameter:

7µm,

30µm,

100nm

Diameter:

100µm

200µm

Fig. 1—ISO-NOPF microsensor with cable

Notes and Warnings

The NO carbon ber microsensors are robust, but not indestructible. Exercise

caution when handling the NO sensor to avoid actions that could damage the tip.

Do not bring the tip into contact with hard surfaces like stir bars. See ”Unpacking”

on page 3.

CAUTION: DO NOT EXPOSE SENSOR TO ORGANIC SOLVENTS.

CAUTION: Carefully read the “Probe Unpacking” instructions (found in the

sealed sensor case) before handling the sensor.

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INSTRUMENT DESCRIPTION

Parts List

After unpacking, verify that there is no visible damage to the sensor. Verify that all

items are included:

(2 or 3) NO microsensors (quantity depends on the type of sensor)

(2 or 3) Sensor Performance Evaluations (each sensor is tested individually at WPI)

(1) Instruction Manual

Unpacking

Upon receipt of these sensors, make a thorough inspection of the contents

and check for possible damage. Missing cartons or obvious damage to cartons

should be noted on the delivery receipt before signing. Concealed damage

should be reported at once to the carrier and an inspection requested. Please

read the section entitled “Claims and Returns” on page 23 of this manual.

Please contact WPI Customer Service if any parts are missing at 941-371-1003 or

[email protected].

The microsensors are shipped in a sealed, rigid plastic, hinged box with foam

padding to protect them from damage during shipment.

To open the package, carefully cut the seals on either side of the sensor box. The

sensors are packaged in foam holders so that their tips are physically isolated from

contact with the container. To remove a microsensor from the package, use the

thumb and index nger of one hand to separate the slit in the foam which contains

the sensor and use the thumb and index nger of the other hand to grasp the

sensor at its midsection and gently remove it from the container.

KEEP THE SENSOR STORAGE BOX and the documentation in a safe place. The test

date and serial number of each sensor is printed on the bottom of its box. Use of

the sensor should begin within 30 days of receipt.

Returns: Do not return any goods to WPI without obtaining prior approval (RMA

# required) and instructions from WPI’s Returns Department. Goods returned

(unauthorized) by collect freight may be refused. If a return shipment is necessary,

use the original container, if possible. If the original container is not available, use

a suitable substitute that is rigid and of adequate size. For further details, please

read the section entitled “Claims and Returns” on page 23 of this manual.

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OPERATING INSTRUCTIONS

Environmental Inuences

There are two environmental parameters to which NO sensors are quite sensitive:

temperature and electrical interference.

Temperature

The background current (and to a lesser degree) the selectivity of the NO sensor

is aected by temperature. This is due to the eects of temperature on the partial

pressure of dissolved NO gas in liquid samples, on the permeability of the coatings

and on the conductivities of various sensor components. It is recommended that

a calibration procedure be performed at the same temperature as the experiment

and that temperature be held constant during NO measurement.

Electrical Interference

External, electrical noise sources (like magnetic stirrers, uorescent lights, MRI

machines, electric motors, computers, pumps and other electrical instruments)

may couple into the sensor signal path electromagnetically and impose

undesirable signals in the output record. The magnitude of this external noise

depends on the environment of the laboratory. If the interference introduced

by the electrical signals in the environment is large, identify the noise source and

remove it. It is also important to ground and shield the system properly.

TIP: Refer to your free radical analyzer manual for proper grounding and shielding

techniques. (In the TBR4100 or Apollo1000 manuals, see “Grounding and Noise

Concerns” in the Operating Instructions section.)

Attaching the Sensor to the Microsensor Handle

Once removed from the package, plug the microsensor into a microsensor cable

(WPI #91580) connected to the free radical analyzer (Fig. 2).* Be very careful that

the sensor tip does not come into contact with anything which could damage

it. The sensor should plug in easily. If you encounter resistance, it is probably

due to misalignment of the sensor plug with the socket connector inside the

microsensor cable. Simply realign the sensor by gently rotating it until it snaps into

place.

*NOTE: Current WPI free radical analyzers include the TBR4100, TBR1025 and

Apollo1000. Apollo4000 is the original 4-channel WPI free radical analyzer.

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Insert sensor connector into microsensor cable

Microsensor Cable (91580)

connector

Fig. 2—NO microsensors may be changed or replaced quickly and easily

Polarizing the Sensor

When a non-polarized microsensor is initially connected to a free radical analyzer,

it may display a high (sometimes o-scale) background current. The polarization

voltage applied by the instrument causes a reduction of the background current

to a stabilized baseline value over time. Set the poise voltage to 865mV. (For the

TBR4100/1025, set the Probe Select dial to NO. ) The amount of time required to

reach a stable baseline current varies for each sensor. New sensors typically take

longer, on the order of several hours.

The Performance Evaluation included with your sensor shows the baseline

current and the sensitivity of your sensor when it was quality tested at WPI.

(In addition, it shows the polarization time of your sensor in the WPI labs.) The

baseline value attainable in your lab may be slightly higher or lower, depending

on the temperature* and composition of the test media. For initial performance

verication of an ISO-NOPF in your lab, WPI recommends using the CuCl2

calibration method (Method 2) described on page 8. Once a stable baseline

current is achieved (usually between 500-8000pA), the microsensor is ready for

use.

*NOTE: The background current of the sensor will usually increase with increasing

temperature of the experiment. Although the sensitivity of the sensor does

not change signicantly within the range 20-37°C, it is recommended that the

calibration procedure be performed at the same temperature as the experiment.

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Calibrating the Sensor

Accurate measurements of NO require an accurate calibration. Three calibration

methods are described in this section.

• Method 1 is based on the decomposition of the S-nitrosothiol NO-donor (SNAP)

using CuCl. The NO liberated from SNAP is used to calibrate the sensor.

• Method 2 is similar to Method 1 using CuCl2 as a catalyst. This convenient

method is the one used in WPI laboratories. Experimentally it has been shown

that CuCl2 is less ecient as a catalyst in the conversion of SNAP to NO. (The

conversion ratio is reduced to approximately 60%.) However, it is technically

easier to accomplish than Method 1, and it provides reliable data.

• Method 3 involves preparing aqueous solutions of NO from saturated NO

solutions prepared with NO gas.

WARNING: THIS METHOD USES NO GAS WHICH CAN BE FATAL IF IT IS

MISHANDLED.

Understanding SNAP

SNAP is a stable NO-containing compound that can be used for quantitative

generation of NO in solution. SNAP decomposes to NO and a disulde byproduct

when dissolved in water. However, the rate of decomposition is very slow. The kinetics

of decomposition for this reagent is a function of several parameters including pH,

presence of a catalyst, temperature and light.

In the procedures described here, SNAP is used in combination with a catalyst,

cuprous (I) chloride (CuCl) or cupric (II) chloride (CuCl2), to generate a known

quantity of NO in solution. Note that this protocol does not investigate the eects

of all parameters involved in SNAP decomposition, nor does it propose a model

by which NO is decomposed. The presented procedures provide an empirical

estimation of the amount of generated NO based on the molarity of a standard

(stock) solution of SNAP under a controlled set of parameters.

NOTE: Remember that most NO probes are sensitive to temperature changes. It

is therefore recommended that the calibration of a NO sensor is performed at the

experimental temperature.

Method 1: SNAP using CuCl

This method of calibration results in the 100% conversion of SNAP to NO. The

amount of NO produced, therefore, is based on the nal concentration of SNAP.

NOTE: The described calibration procedure requires the use of cuprous chloride,

CuCl, where CuCl is the active catalyst for the conversion of SNAP to NO. The

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calibration curve assumes only the presence of CuCl and hence a 100% conversion

eciency of SNAP to NO. However, in the presence of oxygen CuCl is readily

oxidized to CuCl2. This will happen naturally if the compound is exposed to air and/

or there is inadequate storage of CuCl. The oxidation product CuCl2 is much less

ecient at catalyzing the conversion of SNAP to NO, and this would appear during

calibration as an apparent low sensitivity of the electrode to NO. Since CuCl is

readily oxidized to CuCl2 special precautions must be taken to keep it in its reduced

state prior to any calibration. It is recommended that CuCl be stored under inert

conditions, and (if used in solution) the solution must be degassed with inert gas

and absent of all oxygen.

NOTE: If your laboratory is not adequately equipped to satisfy the conditions for

storage and use of CuCl, use the CuCl2 method (Method 2) on page 8.

Preparing Solutions

• Solution #1 (Saturated solution of CuCl): Add 150mg CuCl to 500mL distilled

deoxygenated water. The saturated CuCl solution will have a concentration of

approximately 2.4mM at room temperature and should be kept in the dark prior

to use.

NOTE: The distilled water can be deoxygenated by purging with pure nitrogen or

argon gas for 15 minutes.

• Solution #2 (Standard SNAP solution): Add 5.0mg EDTA to 250mL of water and

deoxygenate the solution. Then add 5.6mg of SNAP and dissolve it completely.

TIP: For complete instructions on making standard 100mM SNAP and calculating

the molarity of SNAP solution, see Appendix A, page 15.

Calibration Procedure

1. Within a nitrogen or argon environment, place 10.0mL of Solution #1 (CuCl) in a

20mL vial.

2. Drop a small stirring bar into the solution, and place the vial on a magnetic stirring

plate.

3. Immerse an NO probe into this solution. While stirring allow the sensor to

polarize until the background current stabilizes. The appropriate time for

stabilization and expected baseline current values depend on the model of the

sensor. (See “Specications” on page 14.)

4. As soon as the background current stabilizes, begin recording the current output.

5. Sequentially inject ve aliquots containing 2µL, 4µL, 8mL, 16mL and 32µL of

Solution #2 (SNAP standard solution) into the vial containing Solution #1 (CuCl).

Immediately following the rst addition of SNAP into Solution#1, the current

(pA) output will increase rapidly. Within a few seconds the response will reach

a plateau. Inject the second aliquot (4µL) as soon as the rst signal reaches a

plateau. Add the third aliquot (8mL) as the second signal reaches its plateau. If

aliquots are not added promptly when reaching the previous plateau, the signal

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will slowly decline because generated NO is quickly oxidized to nitrite and nitrate

which will not be detected by the probe. Add the fourth and fth aliquots in the

same manner.

6. Construct a calibration curve by plotting the signal output (pA) against the

concentration (nM) of SNAP. (See “Appendix B: Constructing a Calibration Curve”

on page 16.)

Method 2: SNAP using CuCl2

Preparing Solutions

• Solution #1(CuCl2 solution): 250mL 0.1M CuCl2 in distilled water

• Solution #2 (Standard SNAP solution): Add 5.0mg EDTA to 250mL of water. Then,

add 5.6mg of SNAP and dissolve it completely.

TIP: For complete instructions on making standard 100mM SNAP and calculating

the molarity of SNAP solution, see Appendix A, page 15.

Calibration Procedure

1. Place 20.0mL of Solution #1(CuCl2) in a 20mL vial.

2. Drop a small stirring bar into the solution, and place the vial on a magnetic stirring

plate.

3. Immerse an NO probe into this solution. While stirring allow the background

current to stabilize. The appropriate time for stabilization depends on the model

of the sensor. (See “Specications” on page 14.)

4. As soon as the background current stabilizes, begin recording the current output.

5. Sequentially inject ve aliquots containing 2µL, 4µL, 8mL, 16mL and 32µL of the

SNAP standard solution (Solution #2) into the vial containing Solution #1 (CuCl2).

Immediately following the rst addition of SNAP into Solution#1, the current

(pA) output will increase rapidly. Within a few seconds the response will reach

a plateau. Inject the second aliquot (4µL) as soon as the rst signal reaches a

plateau. Add the third aliquot (8mL) as the second signal reaches its plateau. If

aliquots are not added promptly when reaching the previous plateau, the signal

will slowly decline because generated NO is quickly oxidized to nitrite and nitrate

which will not be detected by the probe. Add the fourth and fth aliquots in the

same manner.

TIP: You can adjust the volume of injected aliquots according to the concentration

of SNAP stock solution. Decrease the volume of aliquot if the electrode is very

sensitive or increase the volume of aliquot if the electrode is less sensitive.

6. Construct a calibration curve by plotting the signal output (pA) against the

concentration (nM) of SNAP. (See “Appendix B: Constructing a Calibration Curve”

on page 16.)

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Method 3: NO Standards Prepared with NO Gas

This method can be used with all NO sensors and has the advantage of allowing

you to calibrate NO sensors in the same environment in which the experimental

measurements will be made. However, it has the disadvantages of added cost,

inconvenience, and greater hazard to the user. All of these factors must be taken

into consideration.

WARNING: NITRIC OXIDE MUST BE HANDLED ONLY IN A WELL-

VENTILATED AREA, TYPICALLY A LABORATORY FUME HOOD WITH

FORCED VENTILATION. THE U.S. OCCUPATIONAL SAFETY AND HEALTH

ADMINISTRATION HAS SET A TIME-WEIGHTED AVERAGE MAXIMUM NO

VALUE AS 25PPM. THAT IS TO SAY, 25PPM IS CITED AS THE MAXIMUM

CONCENTRATION TO WHICH WORKERS MAY BE CONTINUALLY EXPOSED.

BRIEF INHALATION OF CONCENTRATIONS AS LOW AS 200PPM COULD

PRODUCE DELAYED PULMONARY EDEMA WHICH MAY BE FATAL AFTER AN

ASYMPTOMATIC PERIOD OF UP TO 48 HOURS AFTER THE INITIAL EXPOSURE. IT

IS THEREFORE CRITICAL THAT THE PERSONNEL HANDLING THE GAS BE

THOROUGHLY FAMILIAR WITH THE MATERIAL SAFETY DATA SHEET (MSDS) AND

PROPER HANDLING PROCEDURES. THE PRECAUTIONS RECOMMENDED BY THE

GAS MANUFACTURER MUST BE FOLLOWED.

Fig. 3—Setup for preparing a saturated NO aqueous solution must be in a fume hood

with forced ventilation. Nitric oxide is highly toxic, and it escapes into the atmosphere

during prepation of the standards. NO Lecture bottle (14.2L, 98.5%) can be obtained

from Aldrich.

1. Be certain the fume hood is functioning. Inhalation of NO gas is

potentially fatal. See the WARNING above.

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2. Make sure that all ttings and connections are secure. The tubing to be used

should not be permeable to NO. We recommend Tygon® tubing if a polymer

tubing is to be used. This is permeable to NO, but it has the best performance

compared to other polymer tubing of which we are currently aware. Ideally glass

tubing should be used. If Tygon® tubing is used, note that prolonged exposure

to NO aects its properties. Therefore, it is recommended that the tubing be

inspected frequently and that it be replaced when it appears to be brittle. The

pressure regulator and tee purge adaptor should be stainless steel since nitric

oxide is corrosive.

3. Prepare 100mL of a 10% (by weight) KOH solution and place it in the sidearm

ask (Fig. 3). The ask should be sealed with a stopper through which the tubing

passes by means of a Luer tting to a syringe needle which extends almost to

the bottom of the ask. Tubing is used to connect the side arm of the ask to the

vial containing the water to be equilibrated with NO. The KOH solution is used to

remove other nitrogen oxides from the NO gas.

4. Place 20mL of distilled (preferably deionized) water in a small glass vial. Seal the

vial with a stopper and insert (through the stopper) a long syringe needle which

extends almost to the base of the vial. Connect this syringe needle to the tubing

from the KOH ask, as illustrated. Insert an additional shorter syringe needle

which should not extend into the solution. This acts as a pressure relief

during purging.

5. Place the distilled water vial in an ice-water bath. Reducing the temperature

increases the solubility of NO in solution. Thus, when the solution is used at room

temperature, you will be assured of a saturated NO solution.

6. Purge the system with argon (or nitrogen) gas for a period of 30 minutes at a

moderate ow rate such that the pressure is maintained at a safe level (1-2PSI).

When purging, it should be observed that gas is indeed bubbling through the

KOH solution, as well as the distilled water. After 30 minutes turn o the argon

source and switch the tee purge valve to the correct position for purging with NO

from the lecture bottle.

7. If you are using a pure source, purge the system with NO for 5-10 minutes. (If

the source is not pure, it will take longer.) Again, verify that gas is bubbling in the

solution.

WARNING: NO IS NOW ESCAPING FROM THE PRESSURE RELIEF NEEDLE

IN THE STOPPER OF THE DISTILLED WATER VIAL. IT IS IMPERATIVE

THAT THE FUME HOOD BE RUNNING AT MAXIMUM CAPACITY WITH

THE FRONT PANEL CLOSED (IN THE DOWN POSITION).

8. After the time in step 7 has elapsed, turn o the NO source.

9. Immediately remove the two needles from the distilled water vial.

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10. Set the tee purge valve for purging with argon (or nitrogen) gas, and turn on the

argon source. Purge the system for 5-10 minutes at a moderate ow rate. Gas

should be bubbling through the KOH and then escaping from the ask into the

atmosphere. Again, be sure that the fume hood is ventilating well.

11. Turn o the argon (or nitrogen) source, and allow the fume hood to continue to

ventilate for 10-15 minutes to ensure that all traces of NO gas are removed from

the atmosphere.

12. The solution of distilled water should now be saturated with NO. The

concentration of NO produced by this saturation is dependent upon the

temperature. At 0°C, the concentration is approximately 3.3mM, and at 20°C the

concentration is approximately 1.91mM.

13. Dilutions of known concentration can be prepared from this saturated solution.

In preparing a dilution, be careful not to unseal the vial, for this exposes the

solution to atmospheric oxygen.

Once the dilutions are prepared, it is a simple matter to calibrate the instrument.

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INSTRUMENT MAINTENANCE

Storing the Sensor

STANDBY: When not being used for a short period of time (such as overnight),

the microsensor should remain attached to the microsensor cable and kept dry

(not immersed in solution). Before the next experiment, immerse the sensor

in the experimental solution (like, Kreb’s Buer). The background current will

increase until reaching a stable value. Do not be alarmed if the background current

becomes elevated. This is associated with the hydration of the sensor and will not

negatively aect the sensor’s performance.

LONG-TERM: If the microsensor will not be used for more than three or four days,

then it may be stored dry by removing it from the microsensor cable and returning

it to the case in which it was shipped, being very careful to avoid making contact

with the sensor tip.

The NO microsensor is a maintenance-free consumable sensor. When its

performance is no longer satisfactory, remove it from the microsensor cable and

dispose of it, replacing it with a new one.

Cleaning the Sensor

The sensor should be cleaned after each use by suspending the tip in distilled

water for 20-30 minutes to dissolve salts and remove particles which may have

accumulated on the membrane. If the sensor was used in a protein-rich solution,

the tip should rst be soaked in a protease solution for several minutes to remove

protein build-up and then rinsed with distilled water. Enzymatic detergent (Enzol,

WPI #7363-4) can also be used. The sensor can be sterilized chemically using an

appropriate disinfectant (Cidex, WPI #7364). If necessary, gently dab the sensor

with a Kimwipes® to remove residue.

NOTE: ALWAYS rinse with distilled water before storing the sensor dry.

ACCESSORIES

Part Number Description

7363-4 Enzol Enzymatic Detergent, 1 gallon

7364 Cidex Disinfecting Solution

91580 Microsensor Cable

47510 ProGuide Postion/Holder with Base

47520 ProGuide Position/Holder

47530 ProGuide Plus Position/Holder with ne

adjustment

47540 ProGuide Plus with Base

SNAP25 SNAP, 25mg vial

SNAP50 SNAP, 50mg vial

SNAP100 SNAP, 100mg vial

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TROUBLESHOOTING

Issue Possible Cause Solution

Baseline current is below

specied range.

The poise voltage (sensor

setting) may be incorrectly set.

Set the poise voltage to 865mV. (For the

TBR, choosing the NO sensor setting

selects 865mV automatically.) Set the

range at 10nA.

The sensor may be nearing the

end of its usable life.

Perform a 5-point calibration set using

the standard. If the sensor responds

linearly within the desired concentration

range, it is still useable. See “Calibrating

the Sensor” on page 6.

Unstable

baseline

If the baseline hasn’t stabilized

after 2 hours, the polarizing

solution may be contaminated.

Prepare fresh polarizing solution. Use

0.1M CuCl2 only.

External electrical interferences

may be the problem.

Identify and isolate electrical

interferences.

Calibration

dataset is

not linear.

Uneven aliquots may have been

used.

Check the pipetter calibration.

The stock solutions have

deteriorated.

Prepare fresh standard solution. See

“Calibrating the Sensor” on page 6.

Sensitivity

below range

specied

Foreign materials have been

adsorbed on the sensor’s

surface.

If the foreign materials are proteins, use

and enzymatic cleanser like Enzol (WPI

#7363-4) to remove the contaminant.

The sensor has reached end of

its usable life.

Replace the sensor.

NOTE: If you have a problem/issue with your NO Microsensor that falls outside

the denitions of this troubleshooting section, rst perform the CuCl2 Calibration

Procedure (Method 2) exactly as describe on page 8 of this manual and contact

the WPI Technical Support team at 941-371-1003 or [email protected].

14 World Precision Instruments

SPECIFICATIONS

The NO Microsensors conform to the following specications:

NOPF200 NOPF200-L10 NOPF100 NOP70-L NOP3005 NOP3020 NOP30-L NOP007 NOPNM

Outside Diameter (µm) 200 200 100 70 30 30 30 7 Conical

tip:100

Available Length

(mm) 1-5

10 1-5

3 0.5 2 3 2 150mm

Response Time (sec.) < 5 < 5 < 5 < 3 < 3 < 3 < 3 < 3 < 3

Lowest Detection

Limit/Range (nM)

0.2 0.2 0.2 1 1 1 1 0.5 0.5

Nominal Sensitivity-New

Sensor

(pA/nM)

≥20 ≥50 ≥10 ≥10 ≥1≥1.5 ≥1≥1≥0.5

Baseline Drift none none none none none none none none none

Poise Voltage (mV) 865 865 865 865 865 865 865 865 865

Typical Quiescent Base-

line Current, 25ºC (pA)

2500 3500 2000 3500 1000 500 500 300 300

Acceptable Baseline

Range (pA)

500-

8000

500-8000 500-

8000

2000-

6000

150-

3500

500-

5000

20-

6500

200-

1500

50-100

Polarization Time (hrs) 2+ 8+ 2+ 2+ 1+ 1+ 1+ 1+ 1+

1Sensor length varies in 1mm increments (for example, 1mm, 2mm, 3mm...).

2Sensor length is proportional to sensitivity.

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APPENDIX A: PREPARING STOCK SOLUTION

100mM Standard SNAP Solution

SNAP is a green crystalline compound that is sold in 25mg, 50mg and 100mg

vials (WPI # SNAP25, SNAP50, SNAP100). Both the crystalline form and the liquid

solution of SNAP are photo-sensitive and tend to degrade over time. Wrap the

vial of SNAP compound in aluminum foil and store it in the freezer to slow its

degradation. Similarly, store the bottle of SNAP solution in an amber bottle or wrap

it with aluminum foil and store it in the refrigerator.

NOTE: The decomposition of SNAP at low temperature, in the dark and in the

absence of trace metal ions proceeds slowly because of the EDTA (a chelating

reagent).

WPI technicians recommend making fresh standard SNAP solution daily to ensure

accurate calibration of NO sensors. Concentration of SNAP decreases to 5-10% of

its nominal value after approximately 4-5 hours.

To make a 100mM solution of SNAP:

1. Accurately weigh out 5.0mg EDTA (a preservative) and place it in a clean, dry

bottle that will hold at least 250mL.

2. Use a clean, dry 250mL volumetric ask to accurately measure 250mL of HPLC

pure water (HPLC grade, Sigma).

TIP: If your research demands an oxygen-free sample, you can de-oxygenate this

solution by purging it with pure nitrogen or argon gas for 15 minutes.

3. Pour the water into the bottle with EDTA. Replace the cap and shake it for a few

seconds to dissolve the EDTA. It dissolves rapidly.

4. Accurately weight out 5.6mg of crushed, crystalline SNAP.

TIP: Crush any clumps of SNAP powder with a clean instrument like a glass

stirring rod, a popsicle stick or a tooth pick. If you prefer, place the 5.6mg SNAP

on a small piece of lter paper, fold the paper in half and rub it gently between

your ngers to break up any clumps. Be careful not to spill any of the compound.

5. Add the 5.6mg SNAP to the EDTA solution. Verify that none of the green SNAP

compound is left on your lter paper or measuring tray. Replace the cap and

shake it for a few seconds until the green ecks dissolve into solution.

6. Store this standard solution in an amber bottle, if available, or alternatively, wrap

aluminum foil around the bottle to limit light intrusion. This solution should be

refrigerated.

16 World Precision Instruments

The concentration of SNAP (f.w.= 220.3) in the stock solution is calculated as

follows:

[C] = [A•W/(M•V)]1000

[C] = concentration of SNAP (µM)

A = purity of SNAP

W = weight of SNAP (mg)

M = formula weight of SNAP (220.3g/mol)

V = volume of the solution (L)

If SNAP purity is 98.5%, the concentration of standard SNAP stock solution describe

above is:

[C] = [0.985 x 5.6mg/(220.3g/mol x 0.25L)] x 1000 = 100.1µM

NOTE: The purity of SNAP used is extremely important to ensure an accurate

calibration. We recommend the use of high grade SNAP with minimal purity of 98%

or better.

APPENDIX B: CONSTRUCTING A CALIBRATION

CURVE

Because NO sensors can be calibrated in a linear fashion, the magnitude of every

signal should almost double as the volume of SNAP solution added is doubled in

the course of the calibration. The response should be linear from 10 to 1000nM.

Use the recorded data to construct a calibration curve by plotting the signal

output (for example, in pA) against the concentration of SNAP added at that

time. Note that every addition of SNAP solution corresponds with a particular

NO concentration. The sensitivity of the NO probe can be established from the

gradient or slope of the response curve. After the sensitivity of the NO probe is

established, software (like Data-Trax) can be programmed to display data in either

concentration directly (nM or mM) or redox current (pA or nA).

Predicting the Level of Detectable NO CuCl2

Experiments have shown that SNAP is decomposed instantaneously under the

following set of experimental conditions:

• Temperature 25°C

• Catalyst solution 0.1M CuCl2

• SNAP (WPI, 98% purity) - Fresh standard SNAP solution

• CuCl2 is at equilibrium with ambient air (aerobic conditions)

SNAP (RSNO) decomposes to NO and a disulde byproduct according to the

following equation:

2 RSNO➡2NO + RS – SR

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WPI NO Microsensors User manual

Brand: WPI

Model: NO Microsensors

Type: User manual

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WPI NO Microsensors User manual

WPI NO Microsensors User manual

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